Relating the Surface Properties of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) to Their Bactericidal Effect towards a Biofilm of Streptococcus mutans

This study was designed to determine the effects of superparamagnetic iron oxide nanoparticles (SPIONs) on the biological activity of a bacterial biofilm (Streptococcus mutans). Our hypothesis was that the diffusion of the SPIONs into biofilms would depend on their surface properties, which in turn would largely be determined by their surface functionality. Bare, positively charged and negatively charged SPIONs, with hydrodynamic diameters of 14.6 ± 1.4 nm, 20.4 ± 1.3 nm and 21.2 ± 1.6 nm were evaluated. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and electrophoretic mobility (EPM) measurements were used to confirm that carboxylic functional groups predominated on the negatively charged SPIONS, whereas amine functional groups predominated on the positively charged particles. Transmission electron microscopy (TEM) showed the morphology and sizes of SPIONs. Scanning electron microscopy (SEM) and EPM measurements indicated that the surfaces of the SPIONs were covered with biomolecules following their incubation with the biofilm. Bare SPIONs killed bacteria less than the positively charged SPIONs at the highest exposure concentrations, but the toxicity of the bare and positively charged SPIONs was the same for lower SPION concentrations. The positively charged SPIONs were more effective in killing bacteria than the negatively charged ones. Nonetheless, electrophoretic mobilities of all three SPIONs (negative, bare and positively charged) became more negative following incubation with the (negatively-charged) biofilm. Therefore, while the surface charge of SPIONS was important in determining their biological activity, the initial surface charge was not constant in the presence of the biofilm, leading eventually to SPIONS with fairly similar surface charges in situ. The study nonetheless suggests that the surface characteristics of the SPIONS is an important parameter controlling the efficiency of antimicrobial agents. The analysis of the CFU/mL values shows that the SPIONs have the same toxicity on bacteria in solution in comparison with that on the biofilm.     

A Simple and Highly Sensitive Method for Magnetic Nanoparticle Quantitation Using H-NMR Spectroscopy

Iron oxide superparamagnetic nanoparticles (SPIONs) have drawn significant attention because of their potential impact on medical diagnosis and therapy. However, the difficulty of achieving reliable and standardized quantification of these nanoparticles has limited the uniform study of nanoparticle systems. Current measurement techniques have limited sensitivity, and are sophisticated and subject to individual instrumental settings. Here, a characterization method using proton nuclear magnetic resonance (¹H-NMR) spectroscopy is presented that can quantify SPIONs regardless of surface modification. In addition to routine quantification of SPIONs during nanoparticle development, the method can also be used with in vitro nanoparticle assays and potentially with tissue samples for biodistribution studies. Specifically, measurement of water relaxivity shifts (R₁ or R₂) of dissolved SPION samples is correlated with nanoparticle concentration. Unmodified and dextran- and poly(ethylene glycol)-coated SPIONs gave linear correlations between SPION concentration and R₁ and R₂ relaxivities over five orders of magnitude, to below 10 ppb iron. Quantification of SPION concentration was also demonstrated in the presence of RAW 264.7 macrophage cells. A linear correlation between the SPION concentration and relaxivities was observed to <10 ng Fe/mL. This method is a rapid and inexpensive approach for quantitation of SPIONs and exhibits a number of advantages over many of the current methods for quantitative SPION analysis.     

Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.     

Facilitation and inhibition of larval attachment of the bryozoan Bugula neritina in association with mono-species and multi-species biofilms

In this study, we investigated the effect of mono-species and multi-species biofilms on larval attachment of the bryozoan Bugula neritina. The effect of biofilms was examined through a double-dish choice bioassay in which larvae were given the choice of attaching either to a clean surface of a container or to surfaces covered with biofilms. Larvae attached in response to mono-species biofilms of 5 out of 7 bacterial isolates from a subtidal region, but they avoided surfaces covered by biofilms of 7 out of 8 isolates obtained from an intertidal region. In the follow-up choice experiments with multi-species biofilms developed for 2 days, 7 days, 14 days, 28 days and 30 days, larvae preferentially attached to filmed surfaces over the unfilmed surfaces. When biofilms from 2 different tidal regions (intertidal and subtidal) were offered as choices in the double-dish bioassay, larvae in all cases attached on the subtidal biofilms. Two-day-old subtidal biofilms with low densities of bacteria induced significantly higher (p < 0.05) attachment than did 30- day-old intertidal biofilms, which had high bacterial density. Terminal Restriction Fragment Polymorphism (T-RFLP) analysis revealed that the bacterial communities were substantially different in the subtidal and intertidal regions during all periods of the experiment. Attachment of B. neritina on subtidal biofilms did not depend on the bacterial density but rather was negatively correlated with diatom density, thickness of the exopolysaccharide layer and biofilm age. Our results suggest that the larvae of B. neritina can discriminate between biofilmed and clean surfaces and between biofilms developed under different tidal zones.     

Effects of quorum sensing on cell viability in Streptococcus mutans biofilm formation

Streptococcus mutans (S. mutans) uses a quorum sensing (QS) signaling system, which is dependent on competence stimulating peptide (CSP), to regulate diverse physiological activities including bacteriocin production, genetic transformation, and biofilm formation. However, the mechanism of the QS system-induced biofilm formation remains unclear. Here, we demonstrated that the late-stage biofilm formation was increased by the addition of exogenous CSP in S. mutans. The numbers of dead cells in biofilms formed in presence of CSP was 64.5% higher than that without CSP after 12 h (p < 0.05) and 76.3% higher after 24 h (p < 0.05), the numbers of live cells in biofilms formed in presence of CSP were 89.3% higher than that without CSP after 24 h (p < 0.01). The expression of QS-associated genes was increased 3.4-5.3-fold by CSP in biofilms. Our results revealed that cell viability of S. mutans grown in biofilms is affected by the CSP-dependent QS system.     

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe₃O₄) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.     

Integration and Proliferation of Pseudomonas aeruginosa PA01 in Multispecies Biofilms

Despite an increased awareness of biofilm formation by pathogens and the role of biofilms in human infections, the potential role of environmental biofilms as an intermediate stage in the host-to-host cycle is poorly described. To initiate infection, pathogens in biofilms on inanimate environmental surfaces must detach from the biofilm and be transmitted to a susceptible individual in numbers large enough to constitute an infectious dose. Additionally, while detachment has been recognized as a discrete event in the biofilm lifestyle, it has not been studied to the same extent as biofilm development or biofilm physiology. Successful integration of Pseudomonas aeruginosa strain PA01 expressing green fluorescent protein (PA01GFP), employed here as a surrogate pathogen, into multispecies biofilm communities isolated and enriched from sink drains in public washrooms and a hospital intensive care unit is described. Confocal laser scanning microscopy indicated that PA01GFP cells were most frequently located in the deeper layers of the biofilm, near the attachment surface, when introduced into continuous flow cells before or at the same time as the multispecies drain communities. A more random integration pattern was observed when PA01GFP was introduced into established multispecies biofilms. Significant numbers of single PA01GFP cells were continuously released from the biofilms to the bulk liquid environment, regardless of the order of introduction into the flow cell. Challenging the multispecies biofilms containing PA01GFP with sub-lethal concentrations of an antibiotic, chelating agent and shear forces that typically prevail at distances away from the point of treatment showed that environmental biofilms provide a suitable habitat where pathogens are maintained and protected, and from where they are continuously released.     

Effects of flow rate and substrate concentration on the formation and H2 production of photosynthetic bacterial biofilms

Photosynthetic bacteria (PSB), Rhodopseudomonas palustris CQK 01, were immobilized on the surface of a thin glass slide in a lab-scale flat panel photobioreactor under different flow rates and substrate concentrations. The morphology, dry weight and thickness of the mature PSB biofilms were determined to reveal the relationship between biofilm formation and hydrogen production performance. The mature biofilm formed at a low flow rate and a high substrate concentration showed a looser structure, these structures of the mature biofilm then affected the H₂ production performance of the bioreactor during mature stage. The biofilm formed at a flow rate of 228 ml/h and a substrate concentration of 60 mmol/l exhibited the highest dry weight and optimally porous structure, which is beneficial not only for hydrogen removal from the biofilm but also glucose diffusion into the biofilm, thus significantly boosting the photo-hydrogen production performance.     

Biofilm formation by five species of Candida on three clinical materials

Most recalcitrant infections are associated with colonization and microbial biofilm development. These biofilms are difficult to eliminate by the immune response mechanisms and the current antimicrobial. Fungi can form biofilms on biomaterials commonly used in clinical practice (intravascular catheters, dentures, heart valves, implanted devices, contact lenses and other devices) and are associated with infections.A variety of in vitro models using different substrates/devices have been described. These models have been used to investigate the effect of different variables, including flow, growth time, nutrients and physiological conditions on fungal biofilm formation, morphology and architecture.The purpose of our study is to analyze biofilm formation capacity by 84 strains of Candida spp. (23 C. albicans, 23 C. parapsilosis, 16 C. tropicalis, 17 C. glabrata and 5 C. krusei) on three materials used in medical devices and its quantification using a method based on viable cell count.Under the conditions of our study, all assayed Candida strains have been able to form biofilms. All species showed greater biofilm formation capacity on Teflon™, with the exception of C. glabrata which displayed higher biofilm formation capacity on PVC. Biofilm formation by Candida spp. varies depending on the type of material on which it grows and on the species and strain of Candida.The method we propose could be of great use to deepen scientific knowledge on this subject of remarkable clinical significance, considering the absence of standard biofilm formation and quantification techniques on the catheters and the level of difficulty associated to those available.     

Differential biofilms characteristics of Shewanella decolorationis microbial fuel cells under open and closed circuit conditions

Biofilms formation capacities of Shewanella species in microbial fuel cells (MFCs) and their roles in current generation have been documented to be species-dependent. Understandings of the biofilms growth and metabolism are essential to optimize the current generation of MFCs. Shewanella decolorationis S12 was used in both closed-circuit and open-circuit MFCs in this study. The anodic S. decolorationis S12 biofilms could generate fivefold more current than the planktonic cells, playing a dominant role in current generation. Anodic biofilms viability was sustained at 98 ± 1.2% in closed-circuit while biofilms viability in open-circuit decreased to 72 ± 7% within 96 h. The unviable domain in open-circuit MFCs biofilms majorly located at the inner layer of biofilm. The decreased biofilms viability in open-circuit MFCs could be recovered by switching into closed-circuit, indicating that the current-generating anode in MFCs could serve as a favorable electron acceptor and provide sufficient energy to support cell growth and metabolism inside biofilms.     

A dynamic in vitro model for evaluating antimicrobial activity against bacterial biofilms using a new device and clinical-used catheters

The activity of daptomycin compared to vancomycin against Staphylococcus epidermidis-biofilms on intravascular catheters has been evaluated using the new Sevilla device that enables to use medical grade-catheters, in an in vitro model that simulates the in vivo conditions. S. epidermidis-biofilms were obtained on polyurethane catheter segments using the Sevilla device linked to a continuous culture system for 24 h. To assess the antimicrobial activity, at this time the continuous culture system was changed to therapeutic antimicrobial concentration solutions for 48 h. At each 24 h interval time, catheter segments were taken out, washed and sonicated. Viable adherent bacteria were determined by agar plating. Data of surviving bacteria numbers attached to the catheter surface obtained with the Sevilla device showed a very good reproducibility. Daptomycin showed a good activity against S. epidermidis-biofilm on polyurethane catheter surface. After 48 h exposure to daptomycin, surviving adherent bacteria were reduced by 4 log compared to the control with no antimicrobial. Using the same model, vancomycin reduced bacterial survival by only 1.3 log. The Sevilla device enables antimicrobial agent activity against bacterial biofilms grown on the external surface of catheters used in clinical practice to be evaluated. The model used replicates as closely as possible the biofilm formed in a highly standardized way. Using this model, daptomycin demonstrates potent in vitro activity against S. epidermidis-biofilm on a polyurethane catheter; this activity was greater than that showed by vancomycin.     

An in vitro urinary tract catheter system to investigate biofilm development in catheter-associated urinary tract infections

Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1 mL min^-1), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105 ± 16 μm up to 246 ± 67 μm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation.     

Fimbria targeting superparamagnetic iron oxide nanoparticles enhance the antimicrobial and antibiofilm activity of ciprofloxacin against quinolone-resistant E. coli

High quinolone resistance of Escherichia coli limits the therapy options for urinary tract infection (UTI). In response to the urgent need for efficient treatment of multidrug-resistant infections, we designed a fimbriae targeting superparamagnetic iron oxide nanoparticle (SPION) delivering ciprofloxacin to ciprofloxacin-resistant E. coli. Bovine serum albumin (BSA) conjugated poly(acrylic acid) (PAA) coated SPIONs (BSA@PAA@SPION) were developed for encapsulation of ciprofloxacin and the nanoparticles were tagged with 4-aminophenyl-α-D-mannopyrannoside (mannoside, Man) to target E. coli fimbriae. Ciprofloxacin-loaded mannoside tagged nanoparticles (Cip-Man-BSA@PAA@SPION) provided high antibacterial activity (97.1 and 97.5%, respectively) with a dose of 32 μg/mL ciprofloxacin against two ciprofloxacin-resistant E. coli isolates. Furthermore, a strong biofilm inhibition (86.9% and 98.5%, respectively) was achieved in the isolates at a dose 16 and 8 times lower than the minimum biofilm eradication concentration (MBEC) of ciprofloxacin. Weaker growth inhibition was observed with untargeted nanoparticles, Cip-BSA@PAA@SPIONs, confirming that targeting E. coli fimbria with mannoside-tagged nanoparticles increases the ciprofloxacin efficiency to treat ciprofloxacin-resistant E. coli. Enhanced killing activity against ciprofloxacin-resistant E. coli planktonic cells and strong growth inhibition of their biofilms suggest that Cip-Man-BSA@PAA@SPION system might be an alternative and/or complementary therapeutic option for the treatment of quinolone-resistant E. coli infections.     

Study of the initial phase of biofilm formation using a biofomic approach

Aims

The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test® and the Crystal violet staining technique.

Methods and results

Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm. The biofilm formed by these strains was detected after only 45 min with the BioFilm Ring Test® and after 6 h with the Crystal violet method. The enumeration of bacteria of the PA01 strain confirmed that, after 30 min, a significant amount of bacteria had attached on the bottom of the culture wells. After 48 h the Crystal violet method detected a biofilm with all strains. The four strains which rapidly formed a biofilm did not differ from the slow-forming strains by their mucoid character or their swarming motility or their synthesis of rhamnose. They showed higher swimming mobility.

Conclusions

Our results show that the BioFilm Ring Test® is a method specially suited for the study of the initial phase of the formation of a biofilm.

Significance and impact of study

The BioFilm Ring Test® is an easy and rapid alternative to the Crystal violet staining and the enumeration methods.     

Reactive Oxygen Species Mediated Bacterial Biofilm Inhibition via Zinc Oxide Nanoparticles and Their Statistical Determination

The formation of bacterial biofilm is a major challenge in clinical applications. The main aim of this study is to describe the synthesis, characterization and biocidal potential of zinc oxide nanoparticles (NPs) against bacterial strain Pseudomonas aeruginosa. These nanoparticles were synthesized via soft chemical solution process in a very short time and their structural properties have been investigated in detail by using X-ray diffraction and transmission electron microscopy measurements. In this work, the potential of synthesized ZnO-NPs (∼10–15 nm) has been assessed in-vitro inhibition of bacteria and the formation of their biofilms was observed using the tissue culture plate assays. The crystal violet staining on biofilm formation and its optical density revealed the effect on biofilm inhibition. The NPs at a concentration of 100 µg/mL significantly inhibited the growth of bacteria and biofilm formation. The biofilm inhibition by ZnO-NPs was also confirmed via bio-transmission electron microscopy (Bio-TEM). The Bio-TEM analysis of ZnO-NPs treated bacteria confirmed the deformation and damage of cells. The bacterial growth in presence of NPs concluded the bactericidal ability of NPs in a concentration dependent manner. It has been speculated that the antibacterial activity of NPs as a surface coating material, could be a feasible approach for controlling the pathogens. Additionally, the obtained bacterial solution data is also in agreement with the results from statistical analytical methods.     

Rapid evaluation of the antibiotic susceptibility of fuel ethanol contaminant biofilms

Bacterial contaminants from commercial fuel ethanol production facilities were previously shown to form biofilms as mixed cultures under laboratory conditions. In this study, a rapid assay was developed to simultaneously compare isolates for their ability to form biofilms as pure cultures. A total of 10 strains were isolated from a dry-grind fuel ethanol plant that routinely doses with virginiamycin. These were identified by sequence analysis as six strains of Lactobacillus fermentum, two strains of L. johnsonii, and one strain each of L. mucosae and L. amylovorus. Isolates exhibited a range of susceptibility to virginiamycin in a planktonic assay, with MIC’s (minimum inhibitory concentration) of ?0.5-16 μg/ml. Even though all strains were isolated from a mixed culture biofilm, they varied greatly in their ability to form biofilms as pure cultures. Surprisingly, growth as biofilms did not appear to provide resistance to virginiamycin, even if biofilms were grown for 144 h prior to antibiotic challenge.     

Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin activity

Due to the refractory nature of pathogenic microbial biofilms, innovative biofilm eradication strategies are constantly being sought. Thus, this study addresses a novel approach to eradicate Pseudomonas aeruginosa biofilms. Magnetic nanoparticles (MNP), ciprofloxacin (Cipro), and magnetic fields were systematically evaluated in vitro for their relative anti-biofilm contributions. Twenty-four-hour biofilms exposed to aerosolized MNPs, Cipro, or a combination of both, were assessed in the presence or absence of magnetic fields (Static one-sided, Static switched, Oscillating, Static + oscillating) using changes in bacterial metabolism, biofilm biomass, and biofilm imaging. The biofilms exposed to magnetic fields alone exhibited significant metabolic and biomass reductions (p < 0.05). When biofilms were treated with a MNP/Cipro combination, the most significant metabolic and biomass reductions were observed when exposed to static switched magnetic fields (p < 0.05). The exposure of P. aeruginosa biofilms to a static switched magnetic field alone, or co-administration with MNP/Cipro/MNP + Cipro appears to be a promising approach to eradicate biofilms of this bacterium.     

A new biofilm-associated colicin with increased efficiency against biofilm bacteria

Formation of bacterial biofilm communities leads to profound physiological modifications and increased physical and metabolic exchanges between bacteria. It was previously shown that bioactive molecules produced within the biofilm environment contribute to bacterial interactions. Here we describe new pore-forming colicin R, specifically produced in biofilms formed by the natural isolate Escherichia coli ROAR029 but that cannot be detected under planktonic culture conditions. We demonstrate that an increased SOS stress response within mature biofilms induces SOS-dependent colicin R expression. We provide evidence that colicin R displays increased activity against E. coli strains that have a reduced lipopolysaccharide length, such as the pathogenic enteroaggregative E. coli LF82 clinical isolate, therefore pointing to lipopolysaccharide size as an important determinant for resistance to colicins. We show that colicin R toxicity toward E. coli LF82 is increased under biofilm conditions compared with planktonic susceptibility and that release of colicin R confers a strong competitive advantage in mixed biofilms by rapidly outcompeting sensitive neighboring bacteria. This work identifies the first biofilm-associated colicin that preferentially targets biofilm bacteria. Furthermore, it indicates that the study of antagonistic molecules produced in biofilm and multispecies contexts could reveal unsuspected, ecologically relevant bacterial interactions influencing population dynamics in natural environments.     

Peppermint (Mentha piperita) inhibits microbial biofilms in vitro

Microbial biofilms have become increasingly problematic in the food processing and medical industries where they cause food and surface contamination. Biofilms have also been implicated as the cause of serious infections in humans as their occurrence makes it difficult to treat common infections and the likelihood of recurrent infections is high. Due to emerging resistance, conventional control methods are fast becoming ineffective. In this study, the use of a selection of commercial plant extracts is investigated. The inhibitory effects of eight herbal extracts on the development of microbial biofilms was investigated against clinical and reference strains of Pseudomonas aeruginosa and Candida albicans. The antimicrobial activity was investigated on the planktonic forms using the minimum inhibitory concentration assay. The extracts that showed the highest antimicrobial activity against the two test organisms were Echinacea angustifolia (cone flower), Mentha piperita (peppermint) and Rosmarinus officinalis (rosemary) with minimum inhibitory concentration values between 0.38 and 2.5 mg/ml. The crystal violet assay was used to assess the effect of pre-treating a surface with plant extracts on cell attachment and the extent of biofilm development following exposure to extracts (biofilm biomass). Most of the extracts reduced microbial colonization by at least 50%. In contrast, preformed biofilms were less responsive to the majority of extracts, thus growth inhibition was more difficult to achieve. Mentha piperita was the only extract that showed some antibiofilm activity against both pathogens.